We also observed a reduction in basal dendritic spine density of

We also observed a reduction in basal dendritic spine density of layer V neurons in the conditional null mutants ( Figure 3F). To address whether this phenotype was specific to neurons of the deep cortical layers, we examined dendritic growth and complexity and spine density in neurons located in the superficial layers of the cortex ( Figures 3G and 3H), and did not observe any significant differences between ShhcKO and control animals. To

evaluate the functional consequences of these anatomical changes in Shh conditional null animals, we performed whole-cell voltage clamp recordings and examined spontaneous miniature excitatory postsynaptic currents (mEPSCs) in layer V and layer II/III pyramidal neurons in acute brain slices from P21–P28 ShhcKO mice and wild-type littermates ( Figure 3I). We found that spontaneous mEPSC frequency of layer V

neurons was decreased in the Shh conditional null animals, Selleck mTOR inhibitor while mEPSC frequency in layer II/III ( Figures 3J and 3K) was not significantly different. We also observed a 1.6-fold increase in the input resistance of conditional mutants, but no change in mEPSC amplitude between the groups, consistent with a decrease in membrane surface area likely attributable to the decrease in see more dendritic growth observed in the Golgi analysis ( Figures S3A–S3E). Together, these results suggest a requirement for cortical Shh for proper neuron growth and cortical circuit development in vivo. We reasoned that if cortical Shh is expressed by layer V corticofugal projection neurons, and a loss of Shh results in a reduction in the number of postsynaptic contacts, then the Shh receptor would be expressed by the presynaptic partners of those layer V neurons. A potential candidate receptor for cortical Shh function is the receptor Boc, a Robo-related why Ig/fibronectin superfamily member that binds directly and with high affinity to Hedgehog family members (Kavran et al., 2010, Okada et al., 2006, Tenzen et al., 2006 and Yao et al., 2006). Boc

is expressed in the developing and adult nervous system and has been shown to be necessary for Shh-mediated guidance of axon projections (Connor et al., 2005, Fabre et al., 2010 and Okada et al., 2006). To determine if Boc could be a candidate to mediate Shh function in cortical circuitry, we visualized Boc expression by RNA in situ hybridization and by reporter activity (Figure S4A). We used tissue harvested from a Boc heterozygous mutant mouse with the Boc locus targeted with a cassette encoding a β-galactosidase-neomycin fusion (b-geo) and human placental alkaline phosphatase (hPLAP) reporter genes using a gene trap method (Okada et al., 2006). We found that Boc is expressed strongly in the cortex in a population of neurons in layers II/III, IV, and Va in adult mice (Figure S4C), whereas a close homolog of Boc, Cdon, is not expressed in these cells (A.O.

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